The overall aim of this proposal is to test whether certain chemical carcinogens preferentially react with putative regulatory regions in chromatin which have "single-stranded" properties or exhibit a distinctly high rate of "breathing." (The term "single-stranded" will be used operationally to describe such conformations in this proposal.) Our long term goal is to see whether such interactions of carcinogens with "single-stranded" DNA structures could play a critical role in chemical carcinogenesis. The single-stranded DNA structures are generally known to be more reactive to chemical carcinogens than double-stranded DNA. We have previously detected "single-stranded" conformation at specific sites in chromatin employing a novel chemical probe called bromoacetaldehyde (BAA) and have shown that such structures correlate with gene expression. BAA is extremely specific for unpaired DNA bases. Also, BAA is a potential chemical carcinogen since its less potent analogue, chloroacetaldehyde, is carcinogenic. For the purpose of studying where (that is, at which DNA sequence) and how such DNA conformation occurs, we will first detect "single-stranded" structures in specific protein:DNA complexes in vitro using BAA as the tool. Next, the features of the DNA sequences which are prone to adopt non-B conformation ("single-stranded" structure) will be studied using supercoiled plasmid DNA. This approach is based on our previous observation that DNA sequences detected by BAA in chromatin have the potential of forming a non-B conformation containing "single-stranded" properties in the purified supercoiled DNA form. In addition to BAA, a well known chemical carcinogen N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF) will be tested to see if it can also recognize a similar conformation. This study will be extended to a cell culture system to map the BAA and N-acetoxy-AAF binding sites in chromatin. Lastly, the effect on gene expression of chemical carcinogen binding to the regulatory region of genes will be studied using a transient expression assay.